CN116121202A - 一种柯萨奇病毒b组1型毒株及应用 - Google Patents
一种柯萨奇病毒b组1型毒株及应用 Download PDFInfo
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Abstract
本发明公开了一种柯萨奇病毒B组1型毒株,毒株命名为KM7‑X29,于2022年3月6日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:V202213,保藏地址为中国.武汉.武汉大学,该毒株利用Vero细胞进行培养,病毒收获后经过灭活、澄清、超滤浓缩、蔗糖密度梯度离心、除菌过滤,得到病毒纯化液,进一步将该纯化液制备成免疫原性良好、遗传稳定性高的柯萨奇病毒B组1型灭活疫苗。
Description
技术领域
本发明涉及生物医药技术领域,更具体地说是涉及一种柯萨奇病毒B组1型毒株及应用。
背景技术
柯萨奇病毒B组1型(coxsackievirus B1,CVB1)属于小核糖核酸病毒科的肠道病毒属,是B组柯萨奇病毒(CVB)的一个主要成员。CVB1感染会引起各种不同程度的疾病或症状,如手足口病(Hand foot mouth disease,HFMD)、脑炎、心肌炎、无菌性脑膜炎甚至死亡。此外,CVB1引起了显著的发病率,在美国疾病控制和预防中心(CDC)报告的前15种肠道病毒血清型中一直排名靠前(CDC2010)。此外,CVB1还与1型糖尿病(T1DM)的发展有关。虽然CVB1已经构成了相当大的公共卫生威胁,但目前还没有批准的疫苗或抗病毒疗法,因此有必要采取不同的策略加快研发预防性疫苗。
目前肠道病毒疫苗仅有针对脊髓灰质炎病毒和手足口病中肠道病毒71型的灭活疫苗,尚无针对CVB的上市疫苗。国外的研究人员报道了几项CVB疫苗的生产和临床前试验:小鼠接种灭活的CVB1疫苗2~3周即产生了强烈的抗CVB1中和抗体反应;CVB1感染可以加速NOD和SOCS-1-Tg小鼠的糖尿病发病,并且CVB1疫苗不仅可以防止CVB感染,而且可以防止CVB1诱导的SOCS-1-Tg小鼠糖尿病的发展。此外,其他形式的CVB1疫苗,如重组亚单位疫苗、DNA疫苗和减毒活疫苗,在小鼠模型中也有不同程度的保护效果。
灭活疫苗没有减毒活疫苗毒力返祖及可引发疫苗相关疾病的缺点,具有较好免疫原性和安全性。尽管国外已经开发CVB1灭活疫苗,但是其流行株与国内流行株的基因型不一致,其对中国流行株保护效率低,故仍有必要继续开发安全有效的适合中国的CVB1灭活疫苗。
因此,如何提供一种新的CVB1毒株,并将其应用于制备灭活疫苗中是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一种柯萨奇病毒B组1型毒株,可应用于制备柯萨奇病毒B组1型灭活疫苗。
为了实现上述目的,本发明采用如下技术方案:
一种柯萨奇病毒B组1型毒株,所述毒株命名为KM7-X29,于2022年3月6日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:V202213,保藏地址为中国.武汉.武汉大学。
作为与上述技术方案相同的发明构思,本发明还请求保护权所述的柯萨奇病毒B组1型毒株在制备柯萨奇病毒B组1型灭活疫苗中的应用。
作为与上述技术方案相同的发明构思,本发明还请求保护一种柯萨奇病毒B组1型灭活疫苗,包括KM7-X29毒株的抗原和铝佐剂。
优选的,铝含量浓度为0.35mg/ml。
作为与上述技术方案相同的发明构思,本发明还请求保护一种柯萨奇病毒B组1型灭活疫苗的制备方法,其特征在于,以毒株KM7-X29制备灭活疫苗。
作为上述技术方案优选的技术方案,制备疫苗具体过程包括下述步骤:
(1)37℃、5%CO2静止培养Vero细胞,直至细胞长成单层,将柯萨奇病毒B组1型毒株KM7-X29以感染复数为0.001~0.1接种到所述细胞培养液中,35℃、5%CO2下培养3-4天直至细胞病变达到90%以上;
(2)收获病变细胞的培养液,获得病毒收获液澄清过滤后,灭活,超滤浓缩获得病毒浓缩液;
(3)对所述病毒浓缩液经密度梯度离心制得病毒纯化液;
(4)向所述病毒纯化液中加入稀释剂和铝佐剂配制得柯萨奇病毒B组1型灭活疫苗;
优选的,柯萨奇病毒B组1型病毒收获液的制备方法为:柯萨奇病毒B组1型病毒接种Vero细胞,接种MOI为:0.001~0.1,培养液为病毒维持液,培养条件为35±1℃、5%CO2,收获时间为2~4天;
优选的,灭活病毒收获液的灭活方法为甲醛灭活,其中,甲醛灭活时间为:37±1℃条件下,灭活7天;
优选的,病毒液超滤浓缩是通过以下方法实现的:
病毒灭活液经切向流膜过滤,浓缩倍数为50-200倍,使用截留分子量为100万,优选超滤浓缩采用孔径为100kD的超滤膜包。
密度梯度离心为:制得的病毒浓缩液先用20%蔗糖垫离心,重悬沉淀,将重悬病毒液经蔗糖梯度(浓度范围:从底层开始2.5ml 60%蔗糖;2.5ml 45%蔗糖;2.5ml 30%蔗糖;2.5ml 15%蔗糖),40000rpm 4℃离心6h,PBS悬浮所得样品。
作为与上述技术方案相同的发明构思,本发明还请求保护所述的柯萨奇病毒B组1型毒株在制备核酸标准品的应用。
作为与上述技术方案相同的发明构思,本发明还请求保护所述的柯萨奇病毒B组1型毒株在制备柯萨奇病毒B组1型抗血清产品中的应用。
作为与上述技术方案相同的发明构思,本发明还请求保护一种柯萨奇病毒B组1型病毒抗血清的制备方法,过程为:对动物注射所述柯萨奇病毒B组1型毒株,收集血清,经过Protein G Sepharose High Performance柱纯化后得到所述抗血清,具体过程为:
(1)首次免疫取KM7-X29毒株与弗氏完全佐剂混合乳化,免疫新西兰大耳兔,100μg/只,皮下多点注射;
(2)间隔14天后进行第二次免疫:取KM7-X29毒株与弗氏不完全佐剂混合乳化,100μg/只,皮下多点注射;
(3)间隔14天后进行第三次免疫:取KM7-X29毒株与弗氏不完全佐剂混合乳化,100μg/只,皮下多点注射;
(4)三免后21天采集血液,10000rpm/min离心后取上清,经过100KD超滤膜浓缩后,使用Protein G Sepharose High Performance柱进行纯化,纯化后即为柯萨奇病毒B组1型兔多克隆抗血清,无菌条件下分装,冻存于-80℃备用。采用间接ELISA法和微量中和试验测定抗血清的抗体效价。
作为与上述技术方案相同的发明构思,本发明还请求保护所述的柯萨奇病毒B组1型毒株在制备柯萨奇病毒B组1型感染动物模型中的应用。
作为与上述技术方案相同的发明构思,本发明还请求保护所述的柯萨奇病毒B组1型毒株在制备预防和/或治疗柯萨奇病毒B组1型型所致疾病产品中的应用。
经由上述的技术方案可知,与现有技术相比,本发明方法中繁殖病毒的细胞基质为Vero细胞,是一种世界卫生组织(WHO)推荐的生产人用疫苗的理想细胞基质。这种细胞基质作为人用疫苗细胞基质是安全可靠的。另外,本发明筛选了一株具有代表性的柯萨奇病毒B组1型毒株KM7-X29,利用该毒株制备的疫苗和柯萨奇病毒B组1型抗血清具有治疗和预防柯萨奇病毒B组1型所致疾病的作用,并且柯萨奇病毒B组1型毒株KM7-X29的母传抗体对新生乳鼠也具有保护作用;利用本发明的CVB1毒株KM7-X29还可以构建稳定且重复性好的柯萨奇病毒B组1型急性感染的动物模型,该模型可为下一步进行药物的抗病毒治疗和评估病毒灭活疫苗的免疫保护效果提供研究工具。
而且,本发明采用柯萨奇病毒B组1型毒株KM7-X29株在Vero细胞中培养,可进行柯萨奇病毒B组1型灭活疫苗的高效生产,为大幅度降低生产成本,制备有效、廉价的柯萨奇病毒B组1型灭活疫苗提供了保障;另外,本发明的疫苗为高纯度灭活疫苗,使柯萨奇病毒B组1型灭活疫苗的安全性、有效性得以保证。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1是本发明中CVB1毒株在Vero细胞上适应增殖示意图;
图2是本发明中CVB1毒株蚀斑筛选示意图;
图3是本发明中使用的CVB1毒株QRT-PCR的扩增曲线和标准曲线示意图;
图4是本发明中CVB1毒株感染新生乳鼠生存曲线;
图5是本发明中CVB1毒株感染新生乳鼠胰腺组织HE染色切片图;
图6是本发明中CVB1毒株感染新生乳鼠胰腺组织IHC切片图;
图7是本发明中CVB1毒株感染新生乳鼠胰腺组织病毒载量图;
图8是本发明中使用甲醛灭活在不同时间下CVB1病毒滴度变化曲线图;
图9是本发明中使用的CVB1毒株KM7-X29株电镜示意图;
图10是本发明中CVB1灭活疫苗免疫雌鼠母传抗体对新生乳鼠的保护作用。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:Vero细胞培养
自液氮中取出Vero细胞迅速放入37℃水浴锅中,使细胞悬液快速融化;然后加入含有10%新生牛血清的MEM或DMEM(中国医学科学院医学生物学研究所中心供应室提供)培养液,调pH至7.2,置37℃、5%CO2培养,次日换液。待长成致密单层后,用PBS(中国医学科学院医学生物学研究所中心供应室提供)清洗细胞面,胰酶(中国医学科学院医学生物学研究所中心供应室提供)消化,加入培养液,以1:4~1:8分种率进行细胞传代,直至获得种毒所需的足够细胞。
实施例2:CVB1病毒的分离和鉴定
将从昆明市妇幼保健院得到的100份手足口病儿童患者粪便,各取1g分别用PBS悬浮至5ml,混匀,用0.22μm滤膜过滤除菌。分别将得到的滤液100μL接种Vero细胞,加入病毒维持液,置35℃、5%CO2培养,筛选出有典型细胞病变,病毒增殖、病变最快的一株KM7-X29毒株,命名为KM7-X29,于2022年3月6日保藏于中国典型培养物保藏中心,保藏编号为CCTCCNO:V202213,保藏地址为中国.武汉.武汉大学,见图1。
将获得的毒株取200μL,放入无RNase的1.5mL EP管中(Axygen Body FluidviralDNA/RNA Miniprep Kit,美国),补加200μL BufferV-L涡旋混匀后,静置5min;补加150μLBufferV-N涡旋混匀后,12,000×g离心5min;转移上清至新的2mL离心管中,加300μL含1%冰乙酸的异丙醇,上下倒置6~8次混匀;将过滤柱置于2mL离心管中,并将步骤3的溶液移至过滤柱中,以6000×g转速离心1min;弃滤液,将无盖提取柱放回2mL离心管中,加入500μLBufferW1A静置1min,12,000×g离心1min;弃滤液,将无盖提取柱放回2mL离心管中,加800μL BufferW2,12,000×g离心1min;弃滤液,将无盖提取柱放回2mL离心管中,12,000×g空离2min;将无盖提取柱放于1.5mL离心管中,在提取柱膜中心加50μL洗脱液并静置1min,12,000×g离心1min,洗脱RNA;将洗脱下来的RNA溶液返回到提取柱中央,静置1min,12,000×g离心1min,从而提高RNA洗脱率。丢弃提取柱,RNA溶液于-80℃冰箱保存。
在根据PrimescriptTM One Step RT PCR KitVer.2(Takara,大连)的操作说明并利用小RNA病毒通用引物“AN88-AN89”对上述提取的病毒RNA进行RT-PCR,反应体系RT-PCR反应条件:50℃逆转录30min;94℃预变性2min;94℃变性30s,52℃复性30s,72℃延伸50s,共35个循环;72℃进一步延伸5min。取PCR产物5μl,用1.0%琼脂糖凝胶电泳鉴定后,送往昆明擎科生物科技有限公司测序(Sanger测序法,ABI373XL测序仪)获得约300nt的VP1序列,通过NCBI中Blast比对从而确定病毒血清型。另外,利用MEGA7.0软件对GenBank数据库中获得的CVB1 VP1序列进行比对,根据保守区域设计引物CVB1-VP1F和CVB1-VP1R,并利用NCBI数据库中Primer-BLAST检测引物特异性。利用CVB1-VP1F和CVB1-VP1R测通CVB1全长VP1序列。利用MEGA7.0软件(邻接法,参数Test ofPhylogeny:Bootstrap method,No.ofBootstrap Replications:1000,Mode:Kimura 2-parametermodel)对本次分离到的CVB1毒株进行系统进化分析。该毒株与近年来中国主要的CVB1流行株一同属于GⅡ基因型。
实施例3:CVB1病毒蚀斑纯化和病毒培养
将在Vero细胞上已适应传代到第3代的KM7-X29病毒收获液进行10倍系列稀释,取10-3、10-4、10-5三个稀释度分别接种单层Vero细胞,37℃、5%CO2吸附1h后,先加入不含新生牛血清的MEM维持液,再加入1.5%甲基纤维素-MEM,置35℃、5%CO2培养,显微镜下观察蚀斑形成。蚀斑形成后,用吸头吸取出蚀斑,随即接种于单层Vero细胞,37℃、5%CO2培养3~4天,待细胞病变后收获病毒,收获病毒继续接种于Vero细胞生长传代。经过三轮的蚀斑纯化后挑取单斑,即为KM7-X29单克隆纯化毒株,见图2。
37℃、5%CO2下静止培养Vero细胞,直至细胞长成单层,将CVB1毒株KM7-X29以感染复数为0.001~0.1接种到上述细胞培养液中,置35℃、5%CO2培养2-4天直至细胞病变达到90%以上;收获病变细胞的培养液,获得病毒收获液。将获得的病毒收获液继续接种Vero细胞进行传代培养,直至病毒滴度和免疫原性稳定后,建立病毒种子库,并取样送中国典型培养物保藏中心(简称CCTCC,地址中国武汉,武汉大学),保藏号为CCTCC NO:V202213,命名为KM7-X29。所建立的病毒种子库保存于中国医学科学院医学生物学研究所-70℃冰箱中备用。
实施例4:CVB1病毒滴度的测定
以不含血清的MEM维持液作为病毒稀释液,将实施例3中获得的病毒种子进行10倍系列稀释,将稀释的毒种接种于长满单层Vero细胞的96孔板中(细胞数为1×106细胞/孔),加入含有5%新生牛血清的MEM培养基,37℃、5%CO2培养7天,显微镜下观察细胞病变。本发明按Spearman-Karber法计算病毒滴度(50%cell culture infectious dose,CCID50)LgCCID50=L+d(S-0.5),L为病毒的最低稀释倍数的对数,d为稀释系数,S为细胞病变比值的和(不包括最低比值)。
结果:病毒滴度为:107CCID50/0.1ml。
实施例5:CVB1病毒中和抗体检测
中和实验方法:病毒感染敏感细胞后,引起细胞形态学变化,出现致细胞病变效应(CPE),特异性中和抗体与病毒结合后,可使病毒颗粒失去感染性,抑制CPE的出现。
a)攻击病毒CCID50滴定和滴度梯度制备
按照上述实例4的方法滴定病毒2-3次,取其平均值,求出每0.05ml中含100CCID50的病毒载量;按照计算好的稀释比例稀释攻击病毒,得到实验所需的病毒总量(100CCID50/0.05ml);取3支小管,每管加病毒稀释液0.9ml;吸0.1ml已经稀释好的攻击病毒液到第一支小管中(即10CCID50/0.05ml),更换吸头混匀,按照此方法依次稀释至1CCID50/0.05ml和0.1CCID50/0.05ml。
b)稀释待检血清
待检血清-20℃冻存备检。取无菌小管若干支置试管架上,每管加稀释液0.3ml,加待检血清0.1ml,盖紧盖子,震摇混匀,即为1:4稀释血清。56℃灭活30min。打开无菌包装96孔组织培养板,每孔加0.05ml稀释液,每份血清标本进行2倍系列稀释,每份血清标本的每个稀释度做两个复孔,同时设置每份待测血清对照孔、细胞对照孔各两孔。
c)病毒中和抗体测定的操作步骤:
稀释好血清的96孔板中分别加入0.05ml攻击病毒(含100CCID50/0.05ml),置37℃、5%CO2孵育2h。取一块新的96孔板,做病毒回滴试验(每次实验都必须做)。孵育完成后,用胰酶消化液消化细胞,制备细胞悬液,调节细胞悬液的浓度为2×105个/ml。待检标本板和病毒回滴板每孔分别加入0.1ml细胞悬液,置35℃、5%CO2培养,7天判定最终结果,并记录病毒滴定结果。注意:如果病毒回滴结果不在32-320CCID50/0.05ml的范围内,则试验无效,需重试。
d)结果判定:
当最高稀释度血清的2孔中有1孔出现细胞病变,另一孔不出现细胞病变,该稀释度的倒数即为该血清标本的中和抗体效价;当高稀释度2孔完全病变,相邻低稀释度2孔完全不病变,则两者平均稀释度的倒数即为该血清标本的中和抗体效价;当两个相邻稀释度血清均出现1孔细胞病变,另1孔不出现细胞病变,则两者平均稀释度的倒数即为该血清标本的中和抗体效价。
实施例6:CVB1 TaqMan一步法RT-qPCR方法的建立及核酸标准品的制备(病毒载量部分)
CVB1 TaqMan一步法RT-qPCR方法的建立(见已发文献)
1)引物和探针设计
利用MEGA7.0软件比对GenBank数据库中获得的CVB1的VP1序列,根据保守区域设计特异性引物(CVB1-qP-F、CVB1-qP-R)和Taqman探针(CVB1-probe),并利用Primer-BLAST工具检测引物和探针序列的特异性。由上海生工生物工程股份有限公司合成引物和探针。其中,Taqman探针5’标记的发光基团和3’标记的淬灭基团分别为FAM和BHQ,引物和探针序列见表1。
表1引物和探针
注:参考序列为KM7-X29。M13F和M13R为通用引物。下划线的序列为T7启动子序列。
2)阳性RNA标准品的建立
利用上述扩增引物进行足够量体系的RT-PCR扩增,进行1%琼脂糖凝胶电泳,鉴定片段及切胶回收纯化。产物进行加“A”尾的反应,连接到pMD18-T载体并转化至E.coil DH5α感受态细胞,平板培养后进行菌落PCR,鉴定阳性菌落。菌落PCR使用引物为M13F和M13R(表1)。摇菌扩增阳性菌落,提质粒,使用HindⅢ内切酶酶切质粒,对线性化质粒进行电泳及切胶回收。体外转录出目的RNA片段,利用紫外分光光度计测定RNA浓度。根据公式计算RNA拷贝数:拷贝数(拷贝/ml)=(X g/ml)/(RNA碱基数×340)×6.02×1023。
3)一步法荧光定量RT-PCR反应体系和条件
反应总体系20μl,包括缓冲液2×One Step RT-PCR BufferⅢ10μl,Ex Taq HS0.4μl,PrimeScript RT Enzyme MixⅡ0.4μl,探针B1-DB-probe 0.8μl,引物CVB1-qPF 1μl,引物CVB1-qPR 1μl,RNA模板5μl,RNase Free H2O 1.41μl。反应条件为42℃5min,95℃10s,另95℃5s,60℃30s共40个循环。每次反应均设置3个孔作为无模板对照(no templatecontrol,NTC)。
4)标准曲线的建立
RNA标准品稀释至初始浓度47ng/μl,对应含量为1011拷贝/μl。利用EASY Dilution对标准品RNA进行10倍系列稀释作为模板,含量分别为1×1011~1×101拷贝/μl。按上述体系和条件进行一步法实时荧光定量RT-PCR反应,每个浓度设置3个复孔。在检测极限范围内,根据反应体系中拷贝数的对数和Ct值生成标准曲线。荧光定量仪为CFX96(BIO-RAD),扩增曲线和标准曲线图由Bio-Rad CFX Manager 3.1软件导出,见图3。
并用本实验室提供的EV71、CVA16、CVA6、CVA10、CVB2、CVB3、CVB4、CVB5和CVB1的RNA为模板,利用上述反应体系及条件对该方法的特异性进行评估。
实施例7:CVB1抗病毒血清的制备及效价测定
1、CVB1兔多克隆抗血清的制备
采用CVB1型KM7-X29毒株对新西兰大耳兔进行免疫。免疫程序:首次免疫取KM7-X29毒株与弗氏完全佐剂混合乳化,免疫新西兰大耳兔,100μg/只,皮下多点注射;首次免疫间隔14天后进行第二次免疫:取KM7-X29毒株与弗氏不完全佐剂混合乳化,100μg/只,皮下多点注射;首次免疫间隔28天后进行第三次免疫:取KM7-X29毒株与弗氏不完全佐剂混合乳化,100μg/只,皮下多点注射;第三次免疫后21天后采集血液,10,000rpm离心后取上清,经过100KD超滤膜浓缩后,经过Protein G Sepharose High Performance柱纯化,纯化后即为CVB1兔多克隆抗血清,无菌条件下分装,冻存于-80℃备用。采用间接ELISA法和微量中和试验测定抗血清的抗体效价。
2、微量细胞中和试验
将步骤1的抗血清用稀释液从1:100起开始进行2倍系列稀释,共稀释10个稀释度,加入96孔板,50μl/孔,每个稀释度设置二个复孔。将实施例2的CVB1毒株KM7-X29病毒液(100CCID50/ml)加入96孔板,50μl/孔。置37℃、5%CO2孵育2h后,加入Vero细胞悬液(细胞浓度为2×105个/ml),100μl/孔。置37℃、5%CO2培养7天,观察细胞病变情况,并采用Reed-Muench法计算中和抗体效价。
结果:计算得到CVB1抗血清的中和抗体效价为6145。
3、间接ELISA法测定抗体的抗CVB1效价
包被:抗原用PB溶液按1:8稀释后,加入96孔板中,100μl/孔,用封口膜封板,包被过夜。
洗板:将抗原倒入废液桶,洗液逐排加到96孔板中,液面不可溢出,防止窜孔造成污染,静置30s。将洗液倒入废液桶,拍干水分。重复洗板三次。
封闭:将封闭液200μl加入96孔板中,用封口膜封板,37℃培养箱中孵育2h。
洗板:将封闭液倒入废液桶,洗液逐排加到96孔板中,液面不可溢出,防止窜孔造成污染,静置30s。将洗液倒入废液桶,拍干水分。重复洗板三次。
加抗体:按顺序加CVB1抗血清100μl,CVB1抗血清从1:100开始2倍系列稀释,并用封口膜封板,37℃培养箱中孵育1h。
洗板:将上清倒入废液桶。洗液逐排加到96孔板中,液面不可溢出,防止窜孔造成污染,静置30s。将洗液倒入废液桶,拍干水分。重复洗板三次。
加酶标抗体:用酶标稀释液稀释酶标抗体,加到96孔板中,100μl/孔,用封口膜封板,37℃培养箱中孵育1h。
洗板:将上清倒入废液桶,洗液逐排加到96孔板中,液面不可溢出,防止窜孔造成污染,静置30s。将洗液倒入废液桶,拍干水分。重复洗板三次。
显色:将显色液逐排加到96孔板中,100μl/孔,用封口膜封板,37℃培养箱中避光显色10min。
终止:将终止液逐排加到96孔板中,50μl/孔,30min内读取OD450值。
结果:CVB1抗血清的抗体效价为192000。
4、CVB1抗血清与其他肠道病毒的交叉中和试验
利用本实验室提供的EV71、CVA16、CVA6、CVA10、CVB2、CVB3、CVB4、CVB5和其他5株CVB1毒株,按步骤2进行中和试验,结果为CVB1抗血清与其他肠道病毒的交叉中抗体效价<4,与其他肠道病毒无交叉反应,但可以很好的中和本发明KM7-X29株和其他CVB1毒株,结果见表2。
表2
实施例8:CVB1动物致病性模型的确定
通过颅腔注射(Intracerebral injection,IC)途径,感染剂量为104.5CCID50/只,探索建立(2、3、5、6日)龄的BALB/c新生乳鼠模型,再通过颅腔注射途径分别用102.5、103.5、104.5CCID50/只三种不同的感染剂量感染3日龄BALB/c新生乳鼠,探索建立BALB/c新生乳鼠CVB1感染模型最佳的接种日龄和感染剂量。最终确定建立CVB1模型的最佳日龄和感染剂量,感染条件。
表3临床评分。
临床评分 | 临床表现 |
0分 | 健康 |
1分 | 嗜睡,精神萎靡 |
2分 | 消瘦 |
3分 | 四肢乏力、毛发稀疏、驼背或无力 |
4分 | 濒死和死亡 |
结果显示,对照组各相同日龄小鼠的体重、生存率及临床得分均无显著差异。实验组2日龄乳鼠经IC感染CVB1毒株KM7-X29的实验组与对照组相比表现出乳鼠出现活力下降、精神萎靡、消瘦、四肢乏力、毛发稀疏、驼背等症状,2~3天后发病,7天内全部死亡。实验组3日龄乳鼠经IC感染CVB1毒株KM7-X29的实验组与对照组相比表现出乳鼠出现活力下降、精神萎靡、消瘦、四肢乏力、毛发稀疏、驼背等症状,2~3天后发病,10天内全部死亡。实验组5日龄乳鼠经IC感染剂量CVB1毒株KM7-X29的实验组与对照组相比表现出乳鼠出现活力下降、精神萎靡、消瘦、四肢乏力、毛发稀疏、驼背等症状,2~4天后发病,13天内全部死亡。实验组6日龄乳鼠经IC感染CVB1毒株KM7-X29的实验组与对照组相比表现出乳鼠前12天出现活力下降、精神萎靡、消瘦、四肢乏力、毛发稀疏、驼背等症状,12天后部分乳鼠逐渐恢复健康等症状,20天最终生存率为50%。因此,选择经IC途径接种3日龄乳鼠作为建立乳鼠感染模型的条件。重复性试验证明在该剂量、该途径条件下接种3日龄乳鼠后临床症状典型,平均临床得分为4,乳鼠感染后7天全部死亡,个体的发病时间和死亡率稳定,有非常高的可重复性。见图4(ABC)。再通过颅腔注射途径分别用102.5、103.5、104.5CCID50/只三种不同的感染剂量感染3日龄BALB/c新生乳鼠。经IC感染,剂量为102.5CCID50/只CVB1毒株KM7-X29的实验组与对照组相比表现出乳鼠出现活力下降、精神萎靡、消瘦、四肢乏力、毛发稀疏、驼背等症状,2~3天后发病,12天内全部死亡。经IC感染剂量为103.5CCID50/只CVB1毒株KM7-X29的实验组与对照组相比表现出乳鼠出现活力下降、精神萎靡、消瘦、四肢乏力、毛发稀疏、驼背等症状,2~3天后发病,12天内全部死亡。经IC感染剂量为104.5CCID50/只CVB1毒株KM7-X29的实验组与对照组相比表现出乳鼠出现活力下降、精神萎靡、消瘦、四肢乏力、毛发稀疏、驼背等症状,2~3天后发病,10天内全部死亡。这表明经IC感染104.5CCID50剂量CVB1毒株KM7-X29较为适合建立3日龄CVB1感染模型。见图4(DEF)
在最终确定建立CVB1感染模型最佳组合条件后,以此条件IC途径接种3日龄乳鼠、剂量为104.5CCID50/只建立乳鼠CVB1感染模型,1~5天内每天处死3只KM7-X29感染乳鼠,取其脑、心脏、肺脏、脾脏、胰腺、上肢肌、上肢肌、肠道和血液;血液常温10,000rpm离心10min后,取上层血浆保存于-20℃;其余脏器置10%中性福尔马林固定48h后,经石蜡包埋制作石蜡切片用以进行HE染色和免疫组织化学(immunohistochemistry,IHC)检测。对于免疫组化检测,石蜡切片经过60℃烤片1h、酒精梯度脱蜡、EDTA抗原修复、脱水,抗体CVB1多克隆抗体(本发明制备后面实例7描述)4℃孵育过夜,室温孵育30min后滴加DAB显色,苏木素反蓝。阴性对照组切片抗体采用阴性血清。显微镜下检测结果。其中,抗体CVB1多克隆抗体为利用CVB1病毒三次免疫8周龄新西兰大耳兔得到的多克隆抗体。(本发明制备实例7描述)。
通过HE染色和IHC对经颅腔注射感染CVB1毒株KM7-X29的3日龄乳鼠的的胰腺进行病理学检查,发现CVB1感染会导致乳鼠胰腺组织广泛可见腺泡细胞坏死,胞核碎裂或溶解,细胞分界不清,伴少量炎性细胞浸润,见图5和图6。进行一步法实时荧光定量RT-PCR反应,每个浓度设置3个复孔,使用(本发明制备实例6描述)制备的核酸标准品,对乳鼠胰腺组织进行病毒载量测定,测定结果显示,攻毒后胰腺组织病毒载量在24h迅速升高,在48h达到105.4拷贝/mg,见图7。
实施例9:CVB1病毒的灭活和灭活验证试验
(1)灭活及灭活验证:按CVB1病毒澄清液体积1:4000的比例加入甲醛溶液,使其理论终浓度为100μg/ml,置37±1℃条件下灭活,在灭活的0h、2h、4h、6h、8h、10h、12h、14h、16h、18h、20h、22h、24h、36h、48h、72h、96h、120h、144h进行取样,取样结束后立即用亚硫酸氢钠溶液中和甲醛,透析结束后,进行病毒滴度测定。对每组灭活的病毒滴度取平均值。根据病毒滴度绘制CVB1病毒灭活动力学曲线(使用甲醛灭活浓度下CVB1病毒滴度变化曲线如图6所示)。根据灭活动力学曲线推算病毒完全灭活所需时间(结果见图6)。由图6可见使用1:4000甲醛溶液在36h内均能将CVB1完全灭活,考虑到灭活疫苗规模化生产病毒灭活的安全性风险,选择1:4000作为甲醛灭活的终浓度,即100μg/ml甲醛终浓度作为灭活剂量,37±1℃灭活7天。用培养CVB1的Vero细胞对灭活的CVB1病毒进行3代盲传,观察细胞病变情况,检测灭活效果。
(2)澄清:将病毒灭活液经1级孔径为0.45μm的滤芯过滤,得到病毒灭活澄清液;
实施例10:CVB1病毒的纯化
将实例9中CVB1病毒灭活澄清液,经切向流膜过滤,浓缩倍数为50-200倍,使用截留分子量为100万。优选超滤浓缩,采用孔径为100kD的超滤膜包进行超滤浓缩。将病毒浓缩液采用本领域中常规的蔗糖密度梯度离心法,将浓缩液先用20%蔗糖垫离心,重悬沉淀,将病毒收获液经蔗糖梯度(浓度范围:从底层开始2ml 60%蔗糖;2ml 45%蔗糖;2ml 30%蔗糖;2ml 15%蔗糖),40000rpm 4℃离心6h,用磷酸盐溶液如PBS悬浮所得样品,由此制得柯萨奇CVB1病毒纯化液。通过电镜观察获得20-30nm大小的病毒颗粒,见图7。
实施例11:CVB1灭活疫苗的制备
将实施例3中获得的病毒种子接种Vero细胞工厂,采用无血清的MEM培养基配制成病毒维液,35℃、5%CO2培养2-4天收获病毒,经澄清过滤后,用终浓度1∶4000甲醛溶液在37℃灭活7天。经超滤浓缩、离心纯化后,加入稀释剂和铝佐剂配制疫苗原液,抗原含量为1-20ug/ml,铝含量浓度为0.35mg/ml。
实施例12:CVB1灭活疫苗免疫原性分析
将按照上述实施例11的方法制备的疫苗抗原免疫BALB/c小鼠,做不同免疫剂量的动态免疫原性分析,疫苗抗原含量为1-20ug/ml,与等体积的铝佐剂混合配制剂量为100U/ml的疫苗原液。免疫程序:3针肌注免疫,0.3ml/只,分别1免后、2免后、3免后采血,分离血清,检测中和抗体。在试验过程中,BALB/c小鼠共分6组,每组6只,对照为铝佐剂和M199稀释液组。
中和抗体测定方案如下:
血清按照1:4,1:8,1:16,1:32,1:64,1:128,1:256,1:1024,1:2048,稀释;阳性对照:兔多抗血清;阴性对照:兔阴性血清。
结果表明:疫苗的量效关系显著,随着剂量提高,中和效价随之提高,中和抗体效价大于1∶256,达到了保护性抗体水平,见表4。
表4不同剂量组的不同免疫程序中和抗体结果
实施例13:CVB1母源抗体对乳鼠的保护作用
为研究母传抗体对新生乳鼠的免疫保护作用,对8周龄雌鼠皮下多点、多次注射实例11制备的CVB1疫苗200μl进行第一次免疫,间隔2周后同样的方式注射实例11制备的CVB1疫苗的灭活病毒液与铝佐剂等体积混合200μl以加强免疫。第一次免疫后将雌鼠和雄鼠交配,第二次免疫后3~5天幼鼠出生。选取3日龄乳鼠实验组(11只/组)。另选取8周龄雌鼠,按照上述方法,将CVB1疫苗与佐剂均替换为M199稀释液,对雌鼠注射,将其孕育的3日龄乳鼠作为对照组(5只/组)。
将各组乳鼠分别经IC途径注射107、104.5、102.5的不同剂量的CVB1毒株KM7-X29株,连续观察14天并记录其体重变化、临床评分(采用表3的临床评分标准进行评分)、生存率,以评价CVB1母传抗体对新生乳鼠感染CVB1的保护作用。实验母鼠组孕育的乳鼠在接种病毒后体重正常增加,无临床症状,健康,最终生存率均为100%。对照未免疫母鼠组孕育的乳鼠在接种病毒后体重几乎没有增加,临床症状明显,第2天开始死亡,第3天全部死亡,最终生存率均为0%(图9)。
数据表明,对照组12天后全部死亡,本发明利用KM7-X29株毒株制备的CVB1疫苗实验组的母传抗体能提供乳鼠对致死剂量不同的毒株CVB1的完全保护能力。
实施例14:CVB1不同代次病毒基因组稳定性及序列比较
病毒通过在Vero细胞传代培养,收获P1代次至P15代次的病毒收获液,病毒基因组的核酸提取按TAKARA MiniBEST病毒基因组RNA提取试剂盒操作手册进行,在根据PrimescriptTM One Step RT PCR Kit Ver.2(Takara,大连)的操作说明并利用小RNA病毒通用引物“AN88-AN89”对上述提取的病毒RNA进行RT-PCR,反应体系RT-PCR反应条件:50℃逆转录30min;94℃预变性2min;94℃变性30s,52℃复性30s,72℃延伸50s,共35个循环;72℃进一步延伸5min。取PCR产物5μl,用1.0%琼脂糖凝胶电泳鉴定后,送往昆明擎科生物科技有限公司测序(Sanger测序法,ABI373XL测序仪)获得约300nt的VP1序列,通过NCBI中Blast比对从而确定病毒血清型。另外,利用MEGA7.0软件对GenBank数据库中获得的CVB1VP1序列进行比对,根据保守区域设计引物CVB1-VP1F和CVB1-VP1R,并利用NCBI数据库中Primer-BLAST检测引物特异性。利用CVB1-VP1F和CVB1-VP1R测通CVB1全长VP1序列。利用MEGA7.0软件(邻接法,参数Test of Phylogeny:Bootstrap method,No.of BootstrapReplications:1000,Mode:Kimura 2-parametermodel)对本次分离到收获P1代次至P15代次的病毒收获液,共比较1~15代病毒的VP1和全基因组序列。见表5和表6。
表5KM7-X29株Vero细胞适应性传代各代次全基因序列核酸序列变化
核苷酸位置 | 对应区段 | P1 | P5 | P10 | P15 |
2692 | VP1 | A | G | A | A |
2698 | VP1 | G | A | A | A |
2906 | VP1 | A | A | C | C |
表6KM7-X29株Vero细胞适应性传代各代次氨基酸序列变化
氨基酸位置 | 对应区段 | P1 | P5 | P10 | P15 |
652 | VP1 | N | D | N | N |
654 | VP1 | E | K | K | K |
723 | VP1 | K | K | T | T |
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
序列表
<110> 中国医学科学院医学生物学研究所
<120> 一种柯萨奇病毒B组1型毒株及应用
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Claims (10)
1.一种柯萨奇病毒B组1型毒株,其特征在于,所述毒株命名为KM7-X29,于2022年3月6日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:V202213,保藏地址为中国.武汉.武汉大学。
2.权利要求1所述的柯萨奇病毒B组1型毒株在制备柯萨奇病毒B组1型灭活疫苗中的应用。
3.一种柯萨奇病毒B组1型毒株灭活疫苗,其特征在于,包括KM7-X29毒株的抗原和铝佐剂。
4.一种柯萨奇病毒B组1型毒株灭活疫苗的制备方法,其特征在于,以毒株KM7-X29制备灭活疫苗。
5.根据权利要求4所述的一种柯萨奇病毒B组1型毒株灭活疫苗的制备方法,其特征在于,包括下述步骤:
(1)37℃、5%CO2静止培养Vero细胞,直至细胞长成单层,将柯萨奇病毒B组1型毒株KM7-X29以感染复数为0.001~0.1接种到所述细胞培养液中,35℃、5%CO2培养3-4d直至细胞病变达到90%以上;
(2)收获病变细胞的培养液,获得病毒收获液灭活后,澄清过滤,超滤浓缩获得病毒浓缩液;
(3)对所述病毒浓缩液经密度梯度离心制得病毒纯化液;
(4)向所述病毒纯化液中加入稀释剂和铝佐剂配制得柯萨奇病毒B组1型灭活疫苗。
6.权利要求1所述的柯萨奇病毒B组1型毒株在制备核酸标准品的应用。
7.权利要求1所述的柯萨奇病毒B组1型毒株在制备柯萨奇病毒B组1型抗血清产品中的应用。
8.一种柯萨奇病毒B组1型抗血清的制备方法,其特征在于,具体过程为:对动物注射所述柯萨奇病毒B组1型毒株,收集血清,经过Protein G Sepharose High Performance柱纯化后得到所述抗血清。
9.权利要求1所述的柯萨奇病毒B组1型毒株在制备柯萨奇病毒B组1型感染动物模型中的应用。
10.权利要求1所述的柯萨奇病毒B组1型毒株在制备预防和/或治疗柯萨奇病毒B组1型所致疾病产品中的应用。
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